Biology subjects

D'Souza, A.

Publications and source records attributed to D'Souza, A..

15 recordsLinked to original sources

bioRxiv2026

Unbiased Long-Read Whole-Genome Sequencing Enables High-Resolution Mapping of Transgene Concatenation and Off-target Genomic Disruption in a Mouse Model

Transgenic mouse models are indispensable for dissecting disease mechanisms; yet, their interpretability is frequently compromised by cryptic genomic alterations introduced during transgenesis. Thus, robust quality control strategies are needed to elucidate integration architecture and evaluate model performance when such unintended events occur. Here, we applied unbiased whole-genome long-read sequencing using the PacBio Revio to investigate a mouse model exhibiting unexpected transgene silencing, originally designed to recapitulate autosomal-dominant hereditary macular dystrophy driven by upregulation of a ZZEF1-ALOX15 fusion gene. Long-read sequencing analysis revealed a [≥]29-kb head-to-tail concatemer containing more than three copies of the transgene vector. Reconstruction of transgene-genome junctions revealed off-target integration of the concatemer into the calcium-sensing receptor gene (Casr), along with exogenous E. coli DNA, that together defined final transgene architecture. 5-methylcytosine profiling identified hypermethylation of the transgene promoter and additional phenotyping indicated disruption of endogenous Casr function resulting from the rearrangement. Our workflow enabled direct detection of transgene concatenation and off-target mapping. These findings establish long-read sequencing as a powerful and scalable quality control standard for genetically engineered animal models, uniquely capable of uncovering hidden genomic complexity, resolving aberrant phenotypes, and enhancing the reliability of in vivo disease modelling.

genomics↗

bioRxiv2026

Resident cardiac macrophages are not required for normal atrioventricular node conduction

Resident cardiac macrophages are understood to facilitate atrioventricular (AV) node conduction because they purportedly couple to AV node myocytes via connexin43 (Cx43) containing gap junctions. We tested this mechanism using biophysical modelling, high-resolution imaging of mouse and human AV conduction tissue, and pharmacological macrophage depletion. In silico, coupling macrophage membrane phenotypes to HCN4+ AV node myocytes imposed an electrotonic load that suppressed pacemaking and promoted conduction slowing, including stable 2:1 block in strand simulations. Anatomically, HCN4-defined components of the mouse AV conduction axis were essentially devoid of Cx43 and overlap of CD68+ macrophages and Cx43 was negligible in both mouse AV node and human penetrating bundle. Finally, near-complete macrophage depletion with CSF1R inhibition (PLX5622) did not alter AV electrical activity in vivo or ex vivo. Together, these data argue against a physiologically relevant role for Cx43-mediated macrophage-myocyte electrical coupling in normal AV node function. HIGHLIGHTSO_LIModelling predicts that AV node automaticity and conduction would be suppressed if macrophages coupled to AV node myocytes C_LIO_LIThe mouse AV conduction axis is essentially devoid of Cx43, currently considered responsible for macrophage-AV node myocyte coupling C_LIO_LIOverlap of macrophages and Cx43 expression is not discernible in the Cx43-expressing human distal AV node C_LIO_LIMacrophage depletion by CSF1R inhibition does not impact AV electrical activity in vivo or ex vivo C_LI

physiology↗

bioRxiv2025

Reorganisation of Cortico-Hippocampal White Matter Pathways in Healthy Ageing: Evidence for Paradoxical Shifts in Structural Connectivity.

The hippocampus plays a central role in episodic memory and has been the focus of extensive research over past decades. A substantial body of work has demonstrated that age-related memory decline is linked to changes in how the hippocampus functionally interacts with distributed brain networks. While functional connectivity changes in ageing are well documented, relatively little is known about alterations in the structural connectivity (SC) of the hippocampus, despite its foundational role in supporting communication across neural systems. In this study, we combined high-quality data from the Human Connectome Project and advanced diffusion-weighted imaging (DWI) methods to investigate age-related changes in hippocampal SC. Using a recently developed tractography pipeline that allows greater anatomical specificity than conventional approaches, we systematically compared connectivity patterns between younger (26-30 years) and older (56-60 years) adults. Results revealed reduced hippocampal SC with the entorhinal cortex and medial parietal cortices in older participants, alongside increased SC with anterior temporal areas. This paradoxical pattern suggests that ageing is associated with both vulnerability and reorganisation of hippocampal networks, with increased hippocampal-temporal connectivity potentially reflecting compensatory plasticity in response to reduced posterior medial connection. These findings provide in vivo evidence of cortico-hippocampal structural reorganisation in late middle age, a critical period when pathological processes such as tau deposition are already detectable in cognitively healthy individuals. More broadly, they demonstrate the power of our anatomically refined tractography pipeline as a proof of concept for detecting subtle, regionally specific changes in hippocampal pathway density. This approach holds promise for charting normative ageing trajectories and identifying early biomarkers of vulnerability and compensation in memory-related networks.

neuroscience↗

bioRxiv2025

Disentangling structure-function relationships between the human hippocampus and the whole brain using track-weighted dynamic functional connectivity.

Understanding how structural and functional connectivity shape hippocampal interactions with the rest of the brain is critical for elucidating its role in cognition. Here, we combine high-resolution diffusion MRI, a novel fibre-tracking pipeline designed to specifically probe anatomical connectivity of the in vivo human hippocampus, and track-weighted dynamic functional connectivity (TW-dFC) to investigate how direct anatomical connections between the hippocampus and the rest of the brain relate to time-varying functional interactions. In Study 1, TW-dFC maps were computed for 10 participants from the Human Connectome Project and subjected to ICA and k-means clustering to derive a data-driven parcellation of the hippocampus based on its structure-function relationships. This revealed circumscribed clusters distributed along anterior-posterior and medial-lateral axes, which broadly aligned with hippocampal subfields. In Study 2, we examined the resting-state functional connectivity profiles of each TW-dFC derived cluster in an independent sample of 100 participants. Each hippocampal cluster displayed distinct patterns of functional connectivity with specific substructures within medial temporal, parietal, frontal and occipital cortices as well as subcortical and cerebellar regions. Our findings demonstrate that TW-dFC provides a powerful framework for anatomically informed functional parcellation of the hippocampus and offers new insights into the structural-functional organisation underlying hippocampal-(sub)cortical interactions. Our approach opens new avenues for probing memory systems in health and their disruption in aging and disease.

neuroscience↗

bioRxiv2025

Regional Vulnerability of Cardiac Chambers to Radiotherapy: A Multi-Omics Perspective

The heart is highly vulnerable to radiotherapy (RT)-induced injury, leading to molecular and structural remodeling collectively termed radiation-induced cardiac toxicity (RICT). Although several biological pathways have been implicated, the regional, cardiac-specific molecular responses to radiation exposure remain incompletely understood. Here, a multi-omics approach was adopted to longitudinally characterise the unique responses to radiation of the heart base (including ventricular base and right atrium), or the heart apex. Ventricular base irradiation induced a cardiomyopathy phenotype, with pronounced molecular perturbations in metabolism and electrical conduction, while changes related to tissue structure were predominant following apex-directed RT. In the right atrium, irradiation drives fibrotic tissue remodelling, leading to an increased propensity for atrial fibrillation, underpinned by changes in sarcomere organisation. This study represents a comprehensive characterisation of differential spatiotemporal radiation effects in the heart and highlights biological and functional pathways that are potentially clinically actionable for cardiac radioprotection and monitoring.

cell biology↗

bioRxiv2025

Endurance exercise remodels pulmonary vein sleeve myocytes and promotes a proarrhythmic atrial substrate

BACKGROUNDAtrial fibrillation (AF) susceptibility is heightened in endurance athletes but the underlying mechanisms are incompletely understood. Because pulmonary vein (PV) myocyte triggers are critical determinants of AF, we investigated PV electrophysiological remodelling in animal models of the athletes heart. METHODSThe following experiments were performed in canines and mice after 16 or 6 weeks, respectively, of daily exercise training (ExT), and compared to sedentary (Sed) controls: ECG recording, echocardiography, pharmacological autonomic block, extrastimulus pacing, multielectrode array mapping, monophasic and intracellular action potential (AP) recording with custom-designed pattern recognition analysis, histology, RNAseq and spatial in situ transcriptomics. RESULTSAF propensity was significantly increased in ExT animals. Mapping studies identified heightened rotational activity in the PV-left atrial (LA) junction of ExT vs. Sed canines in vivo, and enhanced automaticity, triggered activity and AP duration variability ex vivo in ExT canines and mice. Intracellular recordings in mouse PV cardiomyocytes determined at least six AP subtypes with increased frequency of pacemaker-like APs in ExT PV, concomitant with increased expression of pacemaking HCN4, Cav1.3 and Cav3.1 channels. PV spontaneous excitability was also significantly enhanced. Subcellular resolution spatial transcriptomics in mouse PV-LA identified diffuse ion channel remodelling and activation of established AF-promoting pro-inflammatory and pro-fibrotic cytokines and chemokines in ExT PV cardiomyocytes. Conduction slowing in the ExT PV-LA junction was attributable to: gap junction remodelling, reduced Na+ channel expression and increased extracellular matrix deposition with enhanced myofibroblast number and proximity to PV cardiomyocytes. CONCLUSIONSEndurance exercise elicits proarrhythmic electro-anatomical remodelling of the PV-LA junction with enhanced pacemaking ion channel expression and immune-inflammatory pathway activation in PV myocytes as prominent contributors. CLINICAL PERSPECTIVEO_ST_ABSWhat is new?C_ST_ABSO_LIThis work is the first demonstration that endurance training results in proarrhythmic electrophysiological remodelling of PV sleeve myocytes and extracellular matrix deposition in the PV-LA junction. C_LIO_LIWe register electrical and molecular heterogeneity of the PV-LA junction at single cell and subcellular resolution, and for the first time identify the molecular events that underlie increased proarrhythmic activity of the trained PV. These include enhanced pacemaking ion channel expression (e.g., HCN4, Cav1.3, and Cav3.1), pro-inflammatory cytokine activation (e.g., TNF, IL-6), increased myofibroblasts and extracellular matrix deposition. C_LI What are the clinical implications?O_LIWe identify the molecular determinants of PV proarrhythmic activity in the trained heart and present new therapeutic targets for AF prevention in athletes. C_LIO_LIOur findings provide mechanistic rationale for the efficacy of pulmonary vein isolation for AF in athletes. C_LI

physiology↗

bioRxiv2024

STK19 facilitates the clearance of lesion-stalled RNAPII during transcription-coupled DNA repair

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

molecular biology↗

bioRxiv2023

XPA tumor variants lead to defects in NER that sensitize cells to cisplatin

Nucleotide excision repair (NER) neutralizes treatment with platinum (Pt)-based chemotherapy by removing Pt lesions from DNA. Previous study has identified that missense mutation or loss of either of the NER genes Excision Repair Cross Complementation Group 1 and 2 (ERCC1 and ERCC2) leads to improved patient outcomes after treatment with Pt-based chemotherapies. Although most NER gene alterations found in patient tumors are missense mutations, the impact of such mutations in the remaining nearly 20 NER genes is unknown. Towards this goal, we previously developed a machine learning strategy to predict genetic variants in an essential NER scaffold protein, Xeroderma Pigmentosum Complementation Group A (XPA), that disrupt repair activity on a UV-damaged substrate. In this study, we report in-depth analyses of a subset of the predicted NER-deficient XPA variants, including in vitro analyses of purified recombinant protein and cell-based assays to test Pt agent sensitivity in cells and determine mechanisms of NER dysfunction. The most NER deficient variant Y148D had reduced protein stability, weaker DNA binding, disrupted recruitment to damage, and degradation resulting from tumor missense mutation. Our findings demonstrate that tumor mutations in XPA impact cell survival after cisplatin treatment and provide valuable mechanistic insights to further improve variant effect prediction efforts. More broadly, these findings suggest XPA tumor variants should be considered when predicting patient response to Pt-based chemotherapy. SignificanceA destabilized, readily degraded tumor variant identified in the NER scaffold protein XPA sensitizes cells to cisplatin, suggesting that XPA variants can be used to predict response to chemotherapy.

cancer biology↗

bioRxiv2022

Building a Tissue-unbiased Brain Template of Fibre Orientation Distribution and Tractography with Multimodal Registration

A brain template provides a standard space for statistical analysis of brain structure and function. For decades, the T1- and T2-weighted brain templates have been widely used for brain grey matter anatomical and functional analysis. However, T1- and T2-weighted templates provide very limited information about the axonal organization within the white matter. Recent advances in Diffusion MRI have enabled the detailed modelling of the axonal fibre orientation distribution (FOD) in white matter. Therefore, building a FOD template is essential for more robust white matter anatomy related analysis; however, it is important that this template aligns well with the cortical and subcortical structures. From such a FOD template, a tractography template can be also generated by fibre tracking algorithms, which can be used for subsequent applications, such as to perform the joint structural and functional analysis while ensuring rigorous fibre-to-fibre correspondence. In this paper, we explore the potential of generating the FOD template based on multimodal registration, in order to constrain the tempalte unbiased to both white and grey matter. We combine the information from T1-weighted, T2-weighted and Diffusion MRI to generate a coherent transformation for FOD registration and template generation. Our FOD template preserves the structural details at the white-grey matter boundary. To illustrate the benefit of this new approach, the resulting tractography template was used for joint structural-functional connectivity analysis.

neuroscience↗

bioRxiv2022

Two Interaction Surfaces between XPA and RPA Organize the Preincision Complex in Nucleotide Excision Repair

The XPA and RPA proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair pathway. We have previously characterized the two interaction surfaces between XPA and RPA, with the RPA32 and RPA70AB subunits. Here we show that the mutations in the two individual interaction surfaces reduce NER activity in biochemical and cellular systems, and that combining mutations in two domains leads to an additive inhibition of NER, suggesting that they fulfill distinct roles. Our data suggest that the interaction between XPA and RPA32 is important for the initial association of XPA with NER complexes, while the interaction between XPA and RPA70 is needed for structural organization of the complex to license the dual incision reaction. SAXS analysis of complexes of XPA and RPA bound to ss/dsDNA junction substrates reveals the architecture of XPA and RPA in the preincision complex and shows that the two interaction domains between RPA and XPA are located at opposite sides of the two molecules. We propose a structure for the overall NER preincision complex that shows that the two strands of the NER bubble assume a U-shape with the two ss/dsDNA junctions localized in close proximity, with the interaction between XPA and RPA70 as one of the key organizing elements.

biochemistry↗

bioRxiv2021

Anatomical connectivity along the anterior-posterior axis of the human hippocampus: new insights using quantitative fibre-tracking.

The hippocampus supports multiple cognitive functions including episodic memory. Recent work has highlighted functional differences along the anterior-posterior axis of the human hippocampus but the neuroanatomical underpinnings of these differences remain unclear. We leveraged track-density imaging to systematically examine anatomical connectivity between the cortical mantle and the anterior-posterior axis of the in-vivo human hippocampus. We first identified the most highly connected cortical areas and detailed the degree to which they preferentially connect along the anterior-posterior axis of the hippocampus. Then, using a tractography pipeline specifically tailored to measure the location and density of streamline endpoints within the hippocampus, we characterised where, within the hippocampus, these cortical areas preferentially connect. Our results were striking in showing that different parts of the hippocampus preferentially connect with distinct cortical areas. Furthermore, we provide evidence that both gradients and circumscribed areas of dense extrinsic anatomical connectivity exist within the human hippocampus. These findings inform conceptual debates in the field by unveiling how specific regions along the anterior-posterior axis of the hippocampus are associated with different cortical inputs/outputs. Overall, our results represent a major advance in our ability to map the anatomical connectivity of the human hippocampus in-vivo and inform our understanding of the neural architecture of hippocampal dependent memory systems in the human brain. This detailed characterization of how specific portions of the hippocampus anatomically connect with cortical brain regions may promote a better understanding of its role in cognition and we emphasize the importance of considering the hippocampus as a heterogeneous structure.

neuroscience↗

bioRxiv2021

Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family

The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an AAC(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment.

microbiology↗

bioRxiv2021

Engineering genetically-encoded synthetic biomarkers for breath-based cancer detection

Breath analysis holds great promise for rapid, noninvasive early cancer detection; however, clinical implementation is impeded by limited signal from nascent tumors and high background expression by non-malignant tissues. To address this issue, we developed a novel breath-based reporter system for early cancer detection using D-limonene, a volatile organic compound (VOC) from citrus fruit that is not produced in humans, in order to minimize background signal and maximize sensitivity and specificity for cancer detection. We metabolically engineered HeLa human cervical cancer cells to express limonene at levels detectable by mass spectrometry by introducing a single plant gene encoding limonene synthase. To improve limonene production and detection sensitivity twofold, we genetically co-expressed a modified form of a key enzyme in the cholesterol biosynthesis pathway. In a HeLa xenograft tumor mouse model, limonene is a sensitive and specific volatile reporter of tumor presence and growth, permitting detection of tumors as small as 5 mm. Moreover, tumor detection in mice improves proportionally with breath sampling time. By continuously collecting VOCs for 10 hours, we improve sensitivity for cancer detection 100-fold over static headspace sampling methods. Whole-body physiologically-based pharmacokinetic (PBPK) modeling and simulation of tumor-derived limonene predicts detection of tumors as small as 7 mm in humans, equivalent to the detection limit of clinical imaging modalities, such as PET, yet far more economical. Significance StatementWe developed a breath-based reporter system using the plant terpene, D-limonene - a volatile secondary metabolite that gives citrus fruit its characteristic scent but is not produced in human tissues - as a biomarker for early cancer detection. Results from this study could pave the way for in vivo gene delivery and tumor-specific expression of exogenous volatile cancer reporters with broad applicability to the early diagnosis of a wide variety of cancers.

bioengineering↗

bioRxiv2021

Extracellular vesicles Transfer Polarized Mitochondria and Increase Cellular Energetics in Ischemic Endothelial Cells

We have demonstrated, for the first time that microvesicles, a sub-type of extracellular vesicles (EVs) derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. We have also demonstrated that transfer of microvesicles, the larger EV fraction, but not exosomes resulted in increased mitochondrial function in hypoxic endothelial cultures. Gene ontology and pathway enrichment analysis of EVs revealed a very high association to glycolysis-related processes. In comparison to heterotypic macrophage- derived EVs, BEC-derived EVs demonstrated a greater selectivity to transfer mitochondria and increase endothelial cell survival under ischemic conditions. HighlightsO_LIMicrovesicles transfer mitochondria to endothelial cells and brain slice neurons C_LIO_LIMitochondrial transfer increased ATP in ischemic brain endothelial cells (BECs) C_LIO_LITransfer of microvesicles increased mitochondrial function in hypoxic BECs C_LIO_LITransfer of exosomes did not affect mitochondrial function in hypoxic BECs C_LIO_LIHomotypic BEC-derived EVs result in greater ATP levels in the recipient BECs C_LI

bioengineering↗

bioRxiv2020

The schizophrenia-associated variant in SLC39A8 alters N-glycosylation in the mouse brain

A missense mutation (A391T) in the manganese transporter SLC39A8 is strongly associated with schizophrenia in genomic studies, though the molecular connection to the brain remains hypothetical. Human carriers of A391T have reduced serum manganese, altered plasma glycosylation, and brain MRI changes consistent with altered metal transport. Here, using a knock-in mouse model homozygous for A391T, we show that the schizophrenia-associated variant changes protein glycosylation in the brain. N-linked glycosylation was most significantly impaired, with effects differing between regions. RNAseq analysis showed negligible regional variation, consistent with changes in the activity of glycosylation enzymes rather than gene expression. Finally, nearly one third of detected glycoproteins were differentially N-glycosylated in the cortex, including members of several pathways previously implicated in schizophrenia such as cell adhesion molecules and neurotransmitter receptors. These findings provide a mechanistic link between a risk allele and biochemical changes in the brain, furthering our molecular understanding of the pathophysiology of schizophrenia.

neuroscience↗